Purification of vaccinia viruses using hydrophobic interaction chromatography

ABSTRACT

The present invention relates to methods for purification of Vaccinia viruses (VV) and/or Vaccinia virus (VV) particles, which can lead to highly pure and stable virus preparations of predominantly biologically active viruses. The invention encompasses purifying a virus preparation in a sterilized way with high efficiency and desirable yield in terms of purity, biological activity and stability, aspects advantageous for industrial production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/622,563, filed Nov. 20, 2009 now U.S. Pat. No. 8,003,364, which is acontinuation-in-part of U.S. application Ser. No. 12/622,474, filed Nov.20, 2009 now U.S. Pat. No. 8,003,363, which is a continuation-in-part ofU.S. application Ser. No. 12/598,362, filed Oct. 30, 2009 now U.S. Pat.No. 8,012,738, which is the U.S. National Stage of InternationalApplication No. PCT/EP2008/003679 filed May 7, 2008, which claims thebenefit of U.S. Provisional Application No. 60/924,413, filed May 14,2007, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods for purification of Vaccinia viruses(VV) and/or Vaccinia virus (VV) particles.

2. Description of Related Art

Traditionally in medicine, a vector is a living organism that does notcause disease itself, but which spreads infection by “carrying”pathogens (agents that cause disease) from one host to another. Avaccine vector is a weakened or killed version of a virus or bacteriumthat carries an inserted antigen (coding for a protein recognized by thebody as foreign) from a disease-causing agent to the subject beingvaccinated. A vaccine vector delivers the antigen in a natural way intothe body and stimulates the immune system into acting against a “safeinfection.” The immune system is led into generating an immune responseagainst the antigen that protects the vaccinated subject against future“risky infections.”

In vaccine development, a recombinant modified virus can be used as thevehicle or vaccine vector for delivering genetic material to a cell.Once in the cell, genetic information is transcribed and translated intoproteins, including the inserted antigen targeted against a specificdisease. Treatment is successful if the antigen delivered by the vectorinto the cell produces a protein, which induces the body's immuneresponse against the antigen and thereby protects against the disease.

A viral vector can be based on an attenuated virus, which cannotreplicate in the host but is able to introduce and express a foreigngene in the infected cell. The virus or the recombinant virus is therebyable to make a protein and display it to the immune system of the host.Some key features of viral vectors are that they can elicit a stronghumoral (B-cell) and cell-mediated (T-cell) immune response.

Viral vectors are commonly used by researchers to develop vaccines forthe prevention and treatment of infectious diseases and cancer, and ofthese, poxviruses (including canary pox, vaccinia, and fowl pox) are themost common vector vaccine candidates.

Pox viruses are a preferred choice for transfer of genetic material intonew hosts due to the relatively large size of the viral genome (appr.150/200 kb) and because of their ability to replicate in the infectedcell's cytoplasm instead of the nucleus, thereby minimizing the risk ofintegrating genetic material into the genome of the host cell. Of thepox viruses, the vaccinia and variola species are the two best known.The virions of pox viruses are large as compared to most other animalviruses (for more details see Fields et al., eds., Virology, 3^(rd)Edition, Volume 2, Chapter 83, pages 2637 ff).

Variola virus is the cause of smallpox. In contrast to variola virus,vaccinia virus does not normally cause systemic disease inimmune-competent individuals and it has therefore been used as a livevaccine to immunize against smallpox. Successful worldwide vaccinationwith Vaccinia virus culminated in the eradication of smallpox as anatural disease in the 1980s (The global eradication of smallpox. Finalreport of the global commission for the certification of smallpoxeradication; History of Public Health, No. 4, Geneva: World HealthOrganization, 1980). Since then, vaccination has been discontinued formany years, except for people at high risk of poxvirus infections (forexample, laboratory workers). However, there is an increasing fear that,for example, variola causing smallpox may be used as a bio-terrorweapon. Furthermore, there is a risk that other poxviruses such ascowpox, camelpox, and monkeypox may potentially mutate, throughselection mechanisms, and obtain similar phenotypes as variola. Severalgovernments are therefore building up stockpiles of Vaccinia-basedvaccines to be used either pre-exposure (before encounter with variolavirus) or post-exposure (after encounter with variola virus) of apresumed or actual smallpox attack.

Vaccinia virus is highly immune-stimulating and provokes strong B-(humoral) and T-cell mediated immunity to both its own gene products andto any foreign gene product resulting from genes inserted in theVaccinia genome. Vaccinia virus is therefore seen as an ideal vector forvaccines against smallpox and other infectious diseases and cancer inthe form of recombinant vaccines. Most of the recombinant Vacciniaviruses described in the literature are based on the fully replicationcompetent Western Reserve strain of Vaccinia virus. It is known thatthis strain has a high neurovirulence and is thus poorly suited for usein humans and animals (Morita et al. 1987, Vaccine 5, 65-70).

In contrast, the Modified Vaccinia virus Ankara (MVA) is known to beexceptionally safe. MVA has been generated by long-term serial passagesof the Chorioallantois Vaccinia Ankara (CVA) strain of Vaccinia virus onchicken embryo fibroblast (CEF) cells (for review see Mayr, A. et al.1975, Infection 3, 6-14; Swiss Patent No. 568,392). Examples of MVAvirus strains deposited in compliance with the requirements of theBudapest Treaty are strains MVA 572, MVA 575, and MVA-BN® deposited atthe European Collection of Animal Cell Cultures (ECACC), Salisbury (UK)with the deposition numbers ECACC V94012707, ECACC V00120707 and ECACCV00083008, respectively, and described in U.S. Pat. Nos. 7,094,412 and7,189,536.

MVA is distinguished by its great attenuation profile compared to itsprecursor CVA. It has diminished virulence or infectiousness, whilemaintaining good immunogenicity. The MVA virus has been analyzed todetermine alterations in the genome relative to the wild type CVAstrain. Six major deletions of genomic DNA (deletion I, II, III, IV, V,and VI) totaling 31,000 base pairs have been identified (Meyer, H. etal. 1991, J. Gen. Virol. 72, 1031-1038). The resulting MVA virus becameseverely host-cell restricted to avian cells. The excellent propertiesof the MVA strain have been demonstrated in extensive clinical trials(Mayr, A. et al. 1978, Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390;Stickl, H. et al. 1974, Dtsch. med. Wschr. 99, 2386-2392), where MVA 571has been used as a priming vaccine at a low dose prior to theadministration of conventional smallpox vaccine in a two-step programand was without any significant adverse events (SAES) in more than120,000 primary vaccinees in Germany (Stickl, H et al. 1974, Dtsch. med.Wschr. 99, 2386-2392; Mayr et al. 1978, Zbl. Bakt. Hyg. I, Abt. Org. B167, 375-390).

MVA-BN® is a virus used in the manufacturing of a stand-alone thirdgeneration smallpox vaccine. MVA-BN® was developed by further passagesfrom MVA strain 571/572. To date, more than 1500 subjects includingsubjects with atopic dermatitis (AD) and HIV infection have beenvaccinated in clinical trials with MVA-BN® based vaccines.

The renewed interest in smallpox vaccine-campaigns with Vaccinia-basedvaccines has initiated an increased global demand for large-scalesmallpox vaccine production. Furthermore, the use of Vaccinia virus as atool for preparation of recombinant vaccines has additionally createdsignificant industrial interest in methods for manufacturing (growth andpurification) of native Vaccinia viruses and recombinant-modifiedVaccinia viruses.

Viruses used in the manufacturing of vaccines or for diagnostic purposescan be purified in several ways depending on the type of virus.Traditionally, purification of pox viruses including Vaccinia virusesand recombinant-modified Vaccinia viruses has been carried out based onmethods separating molecules by means of their size differences. Toenhance removal of host cell contaminants (e.g. DNA and proteins), inparticular DNA, the primary purification by means of size separation hasbeen supplemented by secondary methods such as enzymatic digestion ofDNA (e.g. Benzonase treatment). Most commonly, the primary purificationof Vaccinia viruses and recombinant-modified Vaccinia viruses has beenperformed by sucrose cushion or sucrose gradient centrifugation atvarious sucrose concentrations. Recently, ultrafiltration has also beenapplied either alone or in combination with sucrose cushion or sucrosegradient purification.

Vaccinia Viruses-based vaccines have in general been manufactured inprimary CEF (Chicken Embryo Fibroblasts) cultures. Vaccines manufacturedin primary CEF cultures are generally considered safe as regardsresidual contaminants. First, it is scientifically unlikely that primarycell cultures from healthy chicken embryos should contain any harmfulcontaminants (proteins, DNA). Second, millions of people have beenvaccinated with vaccines manufactured on CEF cultures without anyadverse effects resulting from the contaminants (CEF proteins and CEFDNA). There is, therefore, no regulatory requirement for the level ofhost cell contaminants in vaccines manufactured in primary CEF cultures,but for each vaccine the manufacturer must document its safety. Theregulatory concern for vaccines manufactured in primary CEF culturesrelates to the risk of adventitious agents (microorganisms (includingbacteria, fungi, mycoplasma/spiroplasma, mycobacteria, rickettsia,viruses, protozoa, parasites, TSE agent) that are inadvertentlyintroduced into the production of a biological product).

In the current methods for purification of Vaccinia viruses,manufactured in primary CEF culture the level of CEF protein may be upto 1 mg/dose and the CEF DNA level may exceed 10 μg/dose of 1×10⁸ asmeasured by the TCID50. These levels are considered acceptable from asafety and regulatory perspective as long as the individual vaccinemanufacturer demonstrates that the levels to be found in the Final DrugProduct (FDP) are safe at the intended human indications. Due to therisk of presence of adventitious agents in vaccines manufactured inprimary cell cultures and the associated need for extensive, expensivebiosafety testing of each vaccine batch manufactured, there is a strongstimulus for the vaccine industry to change to continuous cell lines.Once a continuous cell line has been characterized, the need for testingfor adventitious agents of the production batches is minimal.

However, switch from primary to continuous cell culture for productionof Vaccinia and Vaccinia recombinant vaccines is expected to imposestricter safety and regulatory requirements. In fact, the regulatoryauthorities have proposed new requirements for levels of DNAcontaminants in vaccines manufactured using continuous cell lines (SeeDraft FDA guideline), which may be as low as 10 μg host-cell DNA/dose.To achieve such low level of host cell contaminants, new and improvedmethods for purification are needed.

It appears that vaccinia virions are able to bind to heparin through thesurface protein A27L (Chung et al. 1998, J. Virol. 72, 1577-1585). Atleast three surface proteins A27L (Chung et al., J. Virol.72(2):1577-1585, 1998; Ho et al., Journal of Molecular Biology349(5):1060-1071, 2005; Hsiao et al., J. Virol. 72(10):8374-8379, 1998)D8L (Hsiao et al., J. Virol. 73(10):8750-8761, 1999), and H3L (Lin etal., J. Virol. 74(7):3353-3365, 2000) of the most abundant infectiousform of the Vaccinia virus have been reported to bind toglycosaminoglycans.

Examples of glycosaminoglycans in affinity chromatography applicationsare heparin and heparan sulfate. These are highly charged, linear andsulfated polysaccharides composed of repeating disaccharide unitscontaining an uronic acid (glucuronic or iduronic acid) and anN-sulfated or N-acetylated glucosamine (Ampofo et al., AnalyticalBiochemistry 199(2):249-255, 1991; Nugent, Proceedings of the NationalAcademy of Sciences of the United States of America 97(19):10301-10303,2000; Rabenstein, Nat. Prod. Rep. 19:312-331, 2002).

Cellufine® sulfate and sulfated cellulose membranes are sulfated glucosepolymers. Several studies reported antiviral activities of sulfatedcellulose and sulfated dextran/dextrines (Baba et al., Antimicrob.Agents Chemother. 32(11):1742-1745, 1988; Chattopadhyay et al.,International Journal of Biological Macromolecules 43(4):346-351, 2008;Mitsuya et al., Science 240(4852):646-649, 1988; Piret et al., J. Clin.Microbiol. 38(1):110-119, 2000), as well as the binding of virusparticles to Cellufine® sulfate (O'Neil et al., Bio/Technology11:173-178, 1993; Opitz et al., Biotechnol. and Bioeng.103(6):1144-1154, 2009). The precise interaction between these virusesand sulfated cellulose is currently not fully understood.

It has further been suggested that affinity chromatography (Zahn, A andAllain, J.-P. 2005, J. Gen. Virol. 86, 677-685) may be used as basis forpurification of certain virus preparations. There are several examplesfor the application of ion exchange and affinity membrane adsorbers (MA)for the purification of virus particles like adenoviral vectors (Peixotoet al., Biotechnology Progress 24(6):1290-1296, 2008; Sellick, BioPharmInternational 19(1):31-32, 34, 2006), Aedes aegyptidensonucleosis virus(Enden et al., J Theor Biol 237(3):257-264, 2005), baculovirus (Wu etal., Hum. Gene Ther. 18(7):665-672, 2007), and influenza virus (Kalbfusset al., Journal of Membrane Science 299(1-2):251-260, 2007; Opitz etal., Biotechnol. and Bioeng. 103(6):1144-1154, 2009; and Opitz et al.,Journal of Biotechnology 131(3):309-317, 2007).

For efficient purification of vaccinia virus and recombinant vacciniavirus-based vaccines, some significant challenges need to be overcome.Vaccinia virions are far too large to be effectively loaded ontocommercially available heparin columns, e.g., the Hi-Trap heparin columnfrom Amersham Biosciences used by others (Zahn, A and Allain, J.-P.2005, J. Gen. Virol. 86, 677-685) for lab-scale purification ofHepatitis C and B viruses. The Vaccinia virion volume is approximately125 times larger than Hepatitis virion. (The diameter of the Vacciniavirus is, thus, appr. 250 nm as compared with the hepatitis C and Bvirions diameter being appr. 50 nm). Thus, available matrices as, e.g.,used in the column-based approach may not allow for adequate entrance ofvirions into the matrix, loading of sufficient amounts of virusparticles or sufficiently rapid flow through the column to meet theneeds for industrial scale purification. Zahn and Allain worked withvirus load up to 1×10⁶ in up to 1.0 ml volume. For pilot-scalepurification to achieve sufficient material for early clinical trialsvirus loading capacity higher than 1×10¹¹, preferably up 1×10¹³, involumes higher than 5 L, preferably up to 50 L, is needed. Forindustrial purification of Vaccinia virus loading capacity higher than1×10¹³, preferably higher than 1×10¹⁴ in volumes higher than 300 L,preferably higher than 600 L, is needed.

The large size of the Vaccinia virus may prevent effective steric accessbetween the specific surface proteins of the virions and the ligandimmobilized to the matrix. Currently described lab-scale methods of usefor purification of small virus particles may therefore not beindustrially applicable to purification of Vaccinia virus.

Due to the high number of functional surface molecules interacting withthe ligand used for binding of the Vaccinia virus particles, elution ofbound Vaccinia virus may require more harsh and therefore potentiallydenaturing conditions to elute and recover the Vaccinia virus particlesin a biologically effective form in high yields. The matrix, the liganddesign, the method of ligand immobilization, and the ligand density maytherefore require careful design to mediate an effective binding of theVaccinia virus and to permit an effective elution of biologically activeVaccinia virus particles.

Vaccinia virions are too large to be sterile filtered. The method usedin this invention has therefore been developed by to be applicable foran aseptic industrial-scale manufacturing process in a way ensuring fullcompliance with regulatory requirements regarding sterility of vaccines.In line with the above and for the purpose of this invention, the columnsubstituted with the ligand can be applicable for sterilization-in-placeor can be available as a pre-sterilized unit.

In the past, numerous methods like cesium chloride gradientcentrifugation (Payne and Norrby 1976), sucrose cushion or sucrosegradient centrifugation (Esteban and Metz 1973; Joklik 1962; Madalinskiet al. 1977; Zwartouw et al. 1962), tangential-flow filtration anddiafiltration (Greenberg and Kennedy 2008; Monath et al. 2004), as wellas size exclusion chromatography (Stickl et al. 1970), have beendescribed for the isolation and purification of smallpox virusparticles. Introduction of cell culture-derived smallpox vaccinesproduction processes led to a reconsideration of the classicpurification schemes.

Current smallpox vaccines are purified mainly after cell disruption bycentrifugation and filtration methods (Abdalrhman et al. 2006; Greenbergand Kennedy 2008; Monath et al. 2004). However, residual DNA levels needto be further reduced for newly licensed vaccine products fromcontinuous cell lines to comply with current regulations. Accordingly,biopharmaceutical product solutions used for injection should containless than 10 ng of cellular DNA per dose (World-Health-Organization1998) to reduce the possibilities for cellular transformations bypotential oncogenic DNA (Sheng-Fowler et al. 2009) and infections byinfectious DNA. Hence, DNA contaminants need to be reduced, which iscommonly done for smallpox and other vaccines, as well as for viralvectors, by nuclease treatments (Greenberg and Kennedy 2008; Konz et al.2005; Monath et al. 2004; Transfiguracion et al. 2003; Wolff and Reichl2008).

Alternative approaches described in the literature for the clearance ofhost cell DNA from biopharmaceutical products are density gradientcentrifugation, precipitation, anion exchange and affinitychromatography. For example Kumar et al. (Kumar et al. 2002)demonstrated the clearance of host cell DNA from rabies vaccine bydensity gradient centrifugation. Selective precipitation has beendescribed for the preparation of poliovaccines (Amosenko et al. 1991)and recombinant adenoviral vectors (Goerke et al. 2005). Chromatographicapproaches are frequently applied for DNA reductions in recombinantprotein production processes (Gagnon et al. 2006; Knudsen et al. 2001;Sakata and Kunitake 2007; Sakata et al. 2005; Tauer et al. 1995) andviral vaccines (Kalbfuss et al. 2007; Opitz et al. 2009; Opitz et al.2008). Recently, a downstream scheme focusing on a sequentialcombination of pseudo-affinity and anion exchange membrane adsorbers(MA) has been described (Wolff et al. 2009) allowing a significantreduction of DNA in cell culture-derived smallpox vaccines. However, theDNA burden needs to be still improved. Hydrophobic interactionchromatography (HIC) is routinely used in bioseparations (Graumann andEbenbichler 2005; Kramarczyk et al. 2008; Lu et al. 2009; Mahn andAsenjo 2005; Queiroz et al. 2001; Tsumoto et al. 2007; Ueberbacher etal. 2008) since it offers an orthogonal separation technique topurification methods based on ionic interactions. HIC is influenced bymany factors like ligands, ligand densities, applied salts, pH, buffertype and temperature (Graumann and Ebenbichler 2005; Kramarczyk et al.2008; Queiroz et al. 2001).

The influence of salts on hydrophobic interactions follows the lyotropic(Hofmeister) series according to their effect on the solubility ofmacromolecules in aqueous solutions (Graumann and Ebenbichler 2005;Kramarczyk et al. 2008; Queiroz et al. 2001). Antichaotropic salts areconsidered to be water structuring, whereas chaotropic ions randomizeliquid water structure and those are likely to reduce the hydrophobicinteraction strength (Queiroz et al. 2001). In recent years HIC gainedpopularity for the separation of plasmid DNA from impurities like RNA,genomic DNA, lipopolysaccharides and denatured plasmid forms (Diogo etal. 2000).

To achieve a bio-specific purification of Vaccinia virus particles withhigh biological activity, there is a need in the art for development ofindustrially usable ligands for Vaccinia virus purification. Thus, useof a ligand displaying highly specific and highly effective binding tothe Vaccinia virus would be advantageous as it would improvepurification by its ability to specifically sort out biologically activeVaccinia virus particles thereby increasing the purity, viability, andfunctionality of the purified Vaccinia virus.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses methods for virus purification. Theapplication of adsorption chromatography to capture virus particlesafter cell homogenization and cell debris clearance is described. Theinvention includes virus purification using pseudo-affinitychromatography based on heparin and sulfated cellulose and/orhydrophobic interaction chromatography based on ether,poly-propylene-glycol, phenyl, butyl, or hexyl functional groups.

A hydrophobic interaction chromatography media was used to reduce theDNA content of virus preparations. Several different hydrophobicinteraction chromatography ligands were analyzed.

Pseudo-affinity membrane adsorbers, based on reinforced sulfatedcellulose and heparin-membrane adsorbers, were also used. These wereoptimized in terms of dynamic binding capacities and contaminantdepletion

The combination of sulfated cellulose membrane adsorbers with a phenylhydrophobic interaction chromatography resulted in an overall virusrecovery range of 76% to 55%. DNA depletion was reduced to 0.01% of theinitial starting material and the reduction of total protein achieved aprotein contamination below 0.1%.

The invention encompasses methods for purifying biologically activeVaccinia viruses. In one embodiment, the method comprises loading asolid-phase matrix, to which a ligand is attached, with a biologicallyactive Vaccinia virus contained in a liquid-phase culture, washing thematrix; and eluting the biologically active Vaccinia virus.

In one embodiment, the invention encompasses a method for thepurification of biologically active Vaccinia virus comprises binding theVaccinia virus to a solid-phase hydrophobic interaction chromatograpy(HIC) matrix and eluting the biologically active virus. In oneembodiment, the method further comprises binding the Vaccinia virus to asolid-phase pseudo-affinity (PA) matrix; and eluting the biologicallyactive virus.

In one embodiment, the binding the Vaccinia virus to the PA matrix isperformed prior to binding the Vaccinia virus to the HIC matrix. In oneembodiment, the binding the Vaccinia virus to the HIC matrix isperformed prior to binding the Vaccinia virus to the PA matrix.

Preferably, the HIC matrix comprises a PPG ligand, a phenyl ligand, abutyl ligand, or a hexyl ligand.

In one embodiment, the eluted Vaccinia virus contains less than 10 nghost-cell DNA per 10⁸ virus particles. In one embodiment, the methodreduces the amount of dsDNA in the eluted virus to less than 0.04% ofinput. In one embodiment, the method reduces the amount of dsDNA in theeluted virus to less than 0.1% of input.

In one embodiment, the PA matrix comprises or is a membrane. Preferably,the PA matrix comprises or is a sulfated cellulose matrix. Morepreferably, the sulfated cellulose matrix comprises or is a sulfatedreinforced cellulose membrane.

In one embodiment, the PA matrix comprises or is a heparin ligandmembrane.

In one embodiment, the Vaccinia virus is a recombinant Vaccinia virus.In one embodiment, the Vaccinia virus is MVA or recombinant MVA.

In one embodiment, the Vaccinia virus is eluted from the PA matrix withammonium sulfate. In a preferred embodiment, the Vaccinia virus iseluted from the PA matrix with 1.7 M ammonium sulfate.

In one embodiment, the Vaccinia virus is eluted from the HIC matrix witha citric acid buffer.

In one embodiment, the method further comprises a purification step byion-exchange.

In one embodiment, the purified Vaccinia virus retains at least 30% ofits initial TCID50.

In one embodiment, the method further comprises administering the elutedVaccinia virus to an animal, preferably a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts screening of hydrophobic interaction chromatographymedia. Relative amounts of virus (MVA, ELISA), total dsDNA (DNA;Quant-iT® PicoGreen® assay) and total protein (P; Pierce® BCA proteinassay) during the purification of CEF cell-derived MVA-BN® virusparticles using 1 ml columns of ToyoScreen® Ether, ToyoScreen® PPG,ToyoScreen® Phenyl, ToyoScreen® Butyl and ToyoScreen® Hexyl. Adsorptionbuffer: 1.7 M (NH4)2SO4, 50 mM K2HPO4, pH 7.4; elution buffer: 50 mMK2HPO4, pH 7.4. All chromatographic experiments were conducted 3 timesand the individual samples were analyzed as described in the materialand method section; error bars: mean and standard deviation of each testseries.

FIG. 2 depicts purification of MVA-BN® (batch A) by a combination of aSC-MA (15 layers, d=25 mm, A=75 cm²) or heparin-MA (3×15 layers, d=25mm, A=225 cm²) with a 1 ml HIC-phenyl column (ToyoScreen® Phenyl-650M).The loading and equilibration buffer for the SC-MA and heparin-MA was100 mM citric acid, pH 7.4 and the elution buffer 1.7 M (NH₄)₂SO4, 50 mMK₂HPO₄, pH 7.4. The loading and equilibration buffer for the HIC-phenylcolumn corresponded to the elution buffer of the pseudo-affinity MA. Theelution buffer of the HIC-phenyl column was 100 mM citric acid, pH 7.4.The flow rate for the entire process was 1 ml/min. The relative viruscontent was monitored by an ELISA; relative amounts of total protein anddsDNA were quantified by the Pierce® BCA protein assay and the Quant iT™PicoGreen® assay, respectively, based on the initially loaded amounts.All chromatographic experiments were done in triplicates. The ELISA andtotal protein analysis of individual samples were conducted intriplicates and the dsDNA measurements in duplicates.

FIG. 3 depicts determination of the optimal salt concentration forMVA-BN® adsorption to 1 ml ToyoScreen® Phenyl matrix. Relative amountsof MVA-BN® virus particles (ELISA) in the flow through and productfraction as well as total dsDNA (DNA; Quant-iT® PicoGreen®) in the flowthrough and product fraction. Adsorption buffer: 1.7, 1.5, 1.25, 1,0.85, 0.6 and 0.45 M (NH₄)₂SO₄, 50 mM K₂HPO₄, pH 7.4; elution buffer: 50mM K₂HPO₄, pH 7.4. The chromatographic experiments were conducted twiceand individual samples were analyzed in duplicates (dsDNA-assay) andtriplicates (ELISA) as described in the material and method section;error bars: mean and standard deviation of each test series.

DETAILED DESCRIPTION OF THE INVENTION

The invention encompasses methods for purifying viruses. In particular,the present invention is directed to a method for the purification ofbiologically active Vaccinia virus comprising:

a. loading a solid-phase matrix, to which a ligand is attached, with aVaccinia virus contained in a liquid-phase culture;

b. washing the matrix, and

c. eluting the biologically active Vaccinia virus.

The ligand is a substance that, on the one hand, can be attached to thesolid-phase matrix, e.g., by binding or coupling thereto and that, onthe other hand, is able to form a reversible complex with the Vacciniavirus. Thus, by interacting with the virus, the virus is reversiblyretained.

The ligand can be a biological molecule as, for example, a peptideand/or a lectin and/or an antibody and/or, preferably, a carbohydrate.The ligand may also comprise or consist of sulfate. In a furtherembodiment, the ligand comprises one or more negatively charged sulfategroups.

Preferably, the ligand is a hydrophobic molecule as, for example, anaromatic phenyl group, a PPG group, a butyl group, or a hexyl group.

In one embodiment, the method comprises purification of Vaccinia viruswith hydrophobic interaction chromatography (HIC). In a furtherembodiment, the method comprises purification of Vaccinia virus with HICtogether with pseudo-affinity chromatography.

The use of HIC can provide high virus yields with large reductions inDNA and protein contaminants. The level of DNA contamination can bereduced to 0.01% of the initial starting material and the level ofprotein contamination can be reduced to below 0.1%.

Combination of sulfated cellulose based MA with HIC phenyl columnchromatography allowed the purification of CEF cell culture-derivedMVA-BN® virus particles at high virus yields and impressive puritylevels. Protein levels were after SC-MA and HIC-phenyl chromatographypurification independent of the tested production batch below 25 μgtotal protein per dose. Hence, protein levels would be sufficient fornewly licensed cell culture-derived human vaccine products.

Current guidelines for newly licensed human vaccine products fromcontinues cell lines dictate that residual DNA levels exceeding 10 ngper dose are not acceptable (Gijsbers et al. 2005; Sheng-Fowler et al.2009; World-Health-Organization 1998). DNA depletion was, in some cases,sufficient to evade nuclease treatment. A further Benzonase® treatmentcan be included. Due to the tremendously reduced amount of DNA in theproduct fractions, the required amount of Benzonase® for nucleasetreatment can be tremendously reduced allowing a cost-effectivemodification of current downstream processes. Furthermore, a small scaleBenzonase® treatment reduces the probability of vaccines or viralvectors to contain intact oncogenes or further functional DNA sequences.

The invention encompasses a process based on SC-MA in combination withHIC-phenyl chromatography for a downstream process for Vaccinia virusparticles in a manufacturing process and allows for economizing therequired nuclease treatment step compared to classical downstreamprocesses for small pox vaccines or MVA-BN® based viral vectors.

The ligand can be attached to the matrix directly, e.g, by directbinding, or can be attached to the matrix indirectly though anothermolecule, e.g. by coupling through a linker or spacer.

The solid-phase matrix can be a gel, bead, well, membrane, column, etc.In a preferred embodiment of the invention, the solid-phase comprises oris a membrane, in particular a cellulose membrane. However, a broadrange of other polymers modified with specific groups capable to bindthe virus can be used. Preferred are hydrophilic polymers. Examples arecellulose derivatives (cellulose esters and mixtures thereof, cellulosehydrate, cellulose acetate, cellulose nitrate); agarose and itsderivatives; other polysaccharides like chitin and chitosan;polyolefines (polypropylene); polysulfone; polyethersulfone;polystyrene; aromatic and aliphatic polyamides; polysulfonamides;halogenated polymers (polyvinylchloride, polyvinylfluoride,polyvinylidenfluoride); polyesters; homo- and copolymers ofacrylnitrile.

The method and further embodiments of the invention can overcome thelimitations of currently known methods preventing industrial-scale,effective purification of Vaccinia virus particles with high biologicalactivity and purity. The method is superior in terms of yield, processtime, purity, recovery of biologically active Vaccinia virus particlesand costs to existing pilot-scale methods for purification of Vacciniavirus particles, which are primarily based on sucrose-cushioncentrifugation and/or diafiltration or non-specific ion-exchangechromatography. It is also superior in terms of yield, process time,purity, recovery of biologically active Vaccinia virus particles, andcosts to the only existing large-scale method for purification ofVaccinia virus particles, which is based on ultrafiltration, enzymaticDNA degradation, and diafiltration.

According to the present invention, Vaccinia virus can be purified underaseptic conditions to obtain a biologically active, stable, and highlypure virus preparation in high yield. The Vaccinia viruses can be nativeor recombinant.

The present invention provides an improved method for asepticpurification of Vaccinia viruses in lab-, pilot-, and, preferably, inindustrial-scale, leading to a biologically active, stable and highlypure virus preparation in high yield.

This invention provides a more time-effective and cost-effective processfor purification of Vaccinia viruses and recombinant Vaccinia viruses,Modified Vaccinia virus Ankara (MVA) and recombinant MVA, MVA-BN® andrecombinant MVA-BN®, leading to a biologically active, stable and highlypure virus preparation in high yield.

In another embodiment, this invention provides virus preparationsproduced by the method of the invention.

Use of the eluted Vaccinia virus or recombinant Vaccinia virus, orModified Vaccinia virus Ankara (MVA) or recombinant MVA or MVA-BN® orrecombinant MVA-BN®, all preferably obtained by the method according tothe present invention, for the preparation of a pharmaceuticalcomposition, in particular a vaccine, is also an embodiment of theinvention. The virus and/or pharmaceutical preparation is preferablyused for the treatment and/or the prevention of cancer and/or of aninfectious disease.

A method for inducing an immune response or for the vaccination of ananimal, specifically of a mammal, including a human, in need thereof,characterized by the administration of a Vaccinia virus or recombinantVaccinia virus, or Modified Vaccinia virus Ankara (MVA) or recombinantMVA or MVA-BN® or recombinant MVA-BN® vaccine prepared by a processcomprising a purification step as described above is a furtherembodiment of the invention.

As used herein, an “attenuated virus” is a strain of a virus whosepathogenicity has been reduced compared to its precursor, for example byserial passaging and/or by plaque purification on certain cell lines, orby other means, so that it has become less virulent because it does notreplicate, or exhibits very little replication, but is still capable ofinitiating and stimulating a strong immune response equal to that of thenatural virus or stronger, without producing the specific disease.

According to a further preferred embodiment of the present invention,glucosamine glycan (GAG), in particular heparan sulfate or heparin, or aGAG-like substance is used as ligand.

As used herein, “glycosaminoglycans” (GAGs) are long un-branchedpolysaccharides consisting of a repeating disaccharide unit. Some GAGsare located on the cell surface where they regulate a variety ofbiological activities such as developmental processes, bloodcoagulation, tumor metastasis, and virus infection.

As used herein, “GAG-like agents” are defined as any molecule which issimilar to the known GAGs, but can be modified, for example, by theaddition of extra sulfate groups (e.g. over-sulfated heparin). “GAG-likeligands” can be synthetic or naturally occurring substances.Additionally, the term “GAG-like ligands” also covers substancesmimicking the properties of GAGs as ligands in ligand-solid-phasecomplexes. One example for a “GAG-like ligand” mimicking GAG,specifically heparin, as ligand is Sulfate attached to ReinforcedCellulose as solid-phase, thus forming Sulfated Reinforced Cellulose(SRC) as ligand-solid-phase complex. The use of SRC complex is also apreferred embodiment of the present invention. Stabilized ReinforcedCellulose membranes can be obtained, for example, from Sartorius AG.

As used herein, “Bulk Drug Substance” refers to the purified viruspreparation just prior to the step of formulation, fill and finish intothe final vaccine.

As used herein, “Biological activity” is defined as Vaccinia virusvirions that are either 1) infectious in at least one cell type, e.g.CEFs, 2) immunogenic in humans, or 3) both infectious and immunogenic. A“biologically active” Vaccinia virus is one that is either infectious inat least one cell type, e.g. CEFs, or immunogenic in humans, or both. Ina preferred embodiment, the Vaccinia virus is infectious in CEFs and isimmunogenic in humans.

As used herein, “contaminants” cover any unwanted substances which mayoriginate from the host cells used for virus growth (e.g. host cell DNAor protein) or from any additives used during the manufacturing processincluding upstream (e.g. gentamicin) and downstream (e.g. Benzonase).

As used herein, “continuous cell culture (or immortalized cell culture)”describes cells that have been propagated in culture since theestablishment of a primary culture, and they are able to grow andsurvive beyond the natural limit of senescence. Such surviving cells areconsidered as immortal. The term immortalized cells were first appliedfor cancer cells which were able to avoid apoptosis by expressing atelomere-lengthening enzyme. Continuous or immortalized cell lines canbe created, e.g., by induction of oncogenes or by loss of tumorsuppressor genes.

As used herein, “heparan sulfate” is a member of the glycosaminoglycanfamily of carbohydrates. Heparan sulfate is very closely related instructure to heparin, and they both consist of repeating disaccharideunits which are variably sulfated. The most common disaccharide unit inheparan sulfate consists of a glucuronic linked to N-acetyl glucosamine,which typically makes up approx. 50% of the total disaccharide units.

As used herein, “heparin” is a member of the glycosaminoglycan family ofcarbohydrates. Heparin is very closely related in structure to heparansulfate, and they both consist of repeating disaccharide units which arevariably sulfated. In heparin, the most common disaccharide unitconsists of a sulfated iduronic acid linked to a sulfatedglucopyranosyl. To differentiate heparin from heparan sulfate, it hasbeen suggested that in order to qualify a GAG as heparin, the content ofN-sulfate groups should largely exceed that of N-acetyl groups and theconcentration of O-sulfate groups should exceed those of N-sulfate(Gallagher et al. 1985, Biochem. J. 230: 665-674).

As used herein, “industrial scale” or large-scale for the manufacturingof Vaccinia virus or recombinant Vaccinia virus-based vaccines comprisesmethods capable of providing a minimum of 50,000 doses of 1.0×10⁸ virusparticles (total minimum 5.0×10¹² virus particles) per batch (productionrun). Preferably, more than 100,000 doses of 1.0×10⁸ virus particles(total minimum 1.0×10¹³ virus particles) per batch (production run) areprovided.

As used herein, “lab-scale” comprises virus preparation methods ofproviding less than 5,000 doses of 1.0×10⁸ virus particles (total lessthan 5.0×10¹¹ virus particles) per batch (production run).

As used herein, “pilot-scale” comprises virus preparation methods ofproviding more than 5,000 doses of 1.0×10⁸ virus particles (total morethan 5.0×10¹¹ virus particles), but less than 50,000 doses of 1.0×10⁸virus particles (total minimum 5.0×10¹² virus particles) per batch(production run).

As used herein, “Primary cell culture”, refers to the stage where thecells have been isolated from the relevant tissue (e.g. from specificpathogen free (SPF) hens eggs), but before the first sub-culture. Thismeans that the cells have not been grown or divided any further from theoriginal origin.

As used herein, “Purity” of the Vaccinia virus preparation or vaccine isinvestigated in relation to the content of the impurities DNA, protein,Benzonase, and gentamicin. The purity is expressed as specific impurity,which is the amount of each impurity per dose (e.g. ng DNA/dose).

As used herein, “purification” of a Vaccinia virus preparation refers tothe removal or measurable reduction in the level of some contaminant ina Vaccinia virus preparation.

As used herein, “Recombinant Vaccinia virus” is a virus, where a pieceof foreign genetic material (from e.g. HIV virus) has been inserted intothe viral genome. Thereby, both the Vaccinia virus genes and anyinserted genes will be expressed during infection of the Vaccinia virusin the host cell.

As used herein, “Stability” means a measure of how the quality of thevirus preparation (Bulk Drug Substance (BDS) or Final Drug Product(FDP)) varies with time under the influence of a variety ofenvironmental factors such as temperature, humidity and lights, andestablishes a retest period for the BDS or a shelf-life for the FDP atrecommended storage conditions (Guidance for industry Q1A (R2).

As used herein, a “Virus preparation” is a suspension containing virus.The suspension could be from any of the following steps in amanufacturing process: after virus growth, after virus harvest, aftervirus purification (typically the BDS), after formulation, or the finalvaccine (FDP).

As used herein “vaccinia virus forms” refer to the three different typesof virions produced by infected target cells: Mature virions (MV),wrapped virions (WV), and extra-cellular virions (EV) (Moss, B. 2006,Virology, 344:48-54). The EV form comprises the two forms previouslyknown as cell-associated enveloped virus (CEV), and extra-cellularenveloped virus (EEV) (Smith, G. L. 2002, J. Gen. Virol. 83: 2915-2931).

The MV and EV forms are morphologically different since the EV formcontains an additional lipoprotein envelope. Furthermore, these twoforms contain different surface proteins, which are involved in theinfection of the target cells by interaction with surface molecules onthe target cell, such as glycosaminglycans (GAGs) (Carter, G. C. et al.2005, J. Gen. Virol. 86: 12791290). The invention involves use of thepurification of all forms of Vaccinia Virus.

The different forms of Vaccinia virions contain different surfaceproteins, which are involved in the infection of the target cells byinteraction with surface molecules on the target cell, such asglycosaminglycans (GAGs) (Carter, G. C. et al. 2005, J. Gen. Virol. 86:1279-1290). These surface proteins will as mentioned supra be referredto as receptors. On the MV form, a surface protein named p14 or A27L(the latter term will be used in this application) is involved in theinitial attachment of the virions to the target cell. A27L binds to GAGstructures on the target cell prior to entry into the cell (Chung C. etal. 1998, J. Virol. 72: 1577-1585), (Hsiao J. C. et al. 1998 J. Virol.72: 8374-8379), (Vazquez M. et al. 1999, J. Virol. 73: 9098-9109)(Carter G. C. et al. 2005, J. Gen. Virol. 86: 1279-1290). The naturalligand for A27L is presumed to be the GAG known as heparan sulfate (HS).Heparan Sulfate belongs to a group of molecules known asglycosaminglycans (GAGs). GAGs are found ubiquitously on cell surfaces.(Taylor and Drickamer 2006, Introduction to Glycobiology, 2^(nd)edition, Oxford University Press). GAGs are negatively charged moleculescontaining sulfate groups. The A27L protein is located on the surface ofthe virions and is anchored to the membrane by interaction with the A17Lprotein (Rodriguez D. et al. 1993, J. Virol. 67: 3435-3440) (Vazquez M.et al. 1998, J. Virol. 72: 10126-10137). Therefore, the interactionbetween A27L and Al17L can be kept intact during isolation in order toretain full biological activity of the virions. The specific nature ofthe protein-protein interaction between A17L and A27L has not been fullyelucidated, but it has been suggested that a presumed “Leucine-zipper”region in the A27L is involved in the interaction with A17L (Vazquez M.et al. 19981, J. Virol. 72: 10126-10137).

The invention encompasses the use of the affinity interaction betweenthe A27L surface protein on the MV form and glucosaminglycans, inparticular Heparan Sulfate, for purification of the MV form of VacciniaVirus.

The term “ligand”, thus, refers both to a receptor on a target cell andto the specific binding structure attached to a solid-phase matrix usedfor purification of Vaccinia.

The same principle as described above can be applied to interactionsbetween other target cell surface structures and other Vaccinia surfaceproteins of the MV form participating in the Vaccinia virus' recognitionof, attachment to, entry into and/or fusion with the target cell. Theentire A27L protein, or fragments thereof containing the binding regionfor the GAG ligand can be used as agents to elute Vaccinia viruses-GAGcomplexes from a solid-phase column of the invention. Fragments can bereadily generated by routine molecular techniques and screened for theirability to dissociate Vaccinia viruses-GAG complexes using routinetechniques known in the art, such as by measuring eluted, biologicallyactive virus.

The presumed native GAG-ligand for the MV form of Vaccinia is HeparanSulfate (HS) and can be one of the suitable ligands. The invention alsocomprises use of “non-native” ligands for purification of Vacciniavirus. Such non-native ligands are compounds with a high degree ofstructural and/or conformational similarity to native ligands. As anexample, Heparin, which is a close analogue to the native ligand forA27L, HS, can be used for affinity-purification of MV form byinteraction with the A27L surface protein, see further below. Heparinhas been shown to partially inhibit the binding between target cells andVaccinia virus and can therefore also be used for affinity purificationof the MV form of Vaccinia. Other GAG-ligands and GAG-like ligands canalso be used.

In one embodiment of the invention, Heparan Sulfate, used for affinitypurification of the MV form of Vaccinia, binds A27L on biologicallyactive Vaccinia viruses, but does not bind inactive Vaccinia viruses orVaccinia virus fragments.

The purification of Vaccinia virus using HIC allows for a large decreasein the level of cellular DNA contamination of the viral preparation.Thus, a ether, poly-propylene glycol (PPG), phenyl, butyl or hexylligand can be employed.

The ligand makes possible the elution of the bound Vaccinia virus undersuch mild conditions that the Vaccinia virus fully retain theirbiologically activity. This means that virus is infectious, for examplein CEF cells. The infectivity of the Vaccinia virus can be preservedduring purification such that at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the initial TCID₅₀ is retained during purification.Preferably, at least 30% of the initial TCID₅₀ is retained duringpurification. The purification can further comprise a step of binding toa pseudo-affinity (PA) matrix. As used herein, a “pseudo-affinity (PA)matrix” is a solid-phase matrix with an attached pseudo-affinity (PA)ligand. As used herein, a “pseudo-affinity (PA) ligand” is a GAG orGAG-like ligand that binds to Vaccinia virus virions.

The binding and elution characteristics for the GAG-ligand substitutedmatrix depend not only on the individual characteristics of the matrixand ligand, but also on the interplay between the two.

By modifying, e.g., the ligand density or by attaching, e.g. binding orcoupling of, the ligand to the matrix by “arms” or “spacers” ofdifferent length and chemical characteristics (hydrophobicity,hydrophilicity) the binding strength between the target ligand structureand Vaccinia virus can be altered, which can be used to enhance thecapture or ease the elution.

To enhance the purification method, the matrix in the form of achromatography gel or membrane to be used for the purificationpreferably:

-   -   Has a high pore size (to make as many ligands as possible        accessible to the Vaccinia virus)    -   Has a rigid structure to allow for fast flow rates    -   Is available in a form permitting direct or indirect attachment,        e.g. by binding or coupling, of ligands    -   Is applicable for sterilization in place or available as a        pre-sterilized unit, e.g. by using radiation.

In one embodiment, the solid phase matrix is a gel or membrane with apore size of 0.25 μm, preferably of more than 0.25 μm, more preferablyof 1.0-3.0 μm demonstrating a linear flow rate under actual purificationconditions of 10 cm/min, preferably 20 cm/min. The pore size of thematrix can be 0.25-0.5 μm, 0.5-0.75 μm, 0.75-1.0 μm, 1.0-2.0 μm, 2.0-3.0μm, or greater than 3.0 μm.

In one embodiment, with the solid phase matrix containing a heparansulfate as an immobilized ligand, the virus harvest from the upstreamvirus growth process is loaded in a crude (unpurified) form with a flowrate of 10 cm/min, preferably 20 cm/min at a virus concentration of 10⁶virions per mL in pilot scale and 10⁷ virions per mL in industrialscale.

In one embodiment, there are three steps in the purification process ofthe invention, which are common for most affinity chromatographyprocesses:

1) Loading of Vaccinia virus or Vaccinia recombinant virus onto thesolid phase;

2) Washing of the solid phase to remove contaminants; and

3) Elution of the Vaccinia virus or recombinant virus to be isolated.

Step 1. Loading of Vaccinia Virus or Recombinant Virus onto aSolid-Phase Matrix

Loading the solid phase with a ligand can be performed by a batch-,column- or membrane approach.

The membrane approach can have some benefits, specifically for largebio-molecules, in particular for large viruses like Vaccinia viruses:For example, large pore sizes and the availability of the ligand on thesurface of the membrane allow high binding capacities of even largeviral particles. The membrane approach is, thus, a preferred embodimentof the present invention.

In all embodiments mentioned above, the Vaccinia virus or recombinantvirus to be isolated is present in a liquid phase. When the Vacciniavirus or recombinant virus gets close to the ligand, the Vaccinia viruswill bind specifically to or be “captured by” the ligand, thereby theVaccinia virus or recombinant Vaccinia virus can be temporarilyimmobilized on the solid phase, while the contaminants will remain inthe liquid phase.

By appropriate selection of the ligand type, ligand density and ligandsteric configuration, the binding parameters of Vaccinia virus to thesolid phase can be altered, thereby providing means for optimization ofthe purification parameters.

In one embodiment, the virus is bound to the ligand in ammoniumsulphate, for example, at 0.3M, 0.45M, 0.6M, 0.85M, 1.0M, 1.25M, 1.5M,1.7M, 1.85M, or 2.0M.

In various embodiments, the virus is bound to the ligand in citric acid,for example at 100 mM, or with K₂HPO₄, for example at 50 mM at pH7.4. Inpreferred embodiments, the virus is bound in ammonium sulphatecontaining 50 mM K₂HPO₄ at pH7.4.

Step 2. Washing of the Solid Phase to Remove Contaminants

When the binding of the biologically active Vaccinia viruses orrecombinant viruses to the ligand has proceeded sufficiently, the hostcell contaminants (in particular host cell DNA and proteins) that remainin the liquid phase can be removed by washing the solid phase, to whichthe Vaccinia virus is bound, with an appropriate washing medium.

In one embodiment, the solid phase is washed with ammonium sulphate, forexample, at 0.3M, 0.45M, 0.6M, 0.85M, 1.0M, 1.25M, 1.5M, 1.7M, 1.85M, or2.0M.

In various embodiments, the solid phase is washed with citric acid, forexample at 100 mM, or with K₂HPO₄, for example at 50 mM at pH7.4.

Step 3. Eluting the Vaccinia Virus or Recombinant Virus by Specific orNon-Specific Agents

The biologically active Vaccinia viruses or recombinant viruses can beeluted. The elution of the captured Vaccinia virus can be performed, forexample, by:

Agents specifically disrupting the specific interaction between theligand and a L surface protein on the Vaccinia virus (to be calledspecific agents), or by:

Agents non-specifically disrupting the electrostatic interaction betweenthe ligand and the surface protein (to be called non-specific agents).

In one embodiment, the agent is ammonium sulphate, for example, at 0.3M,0.45M, 0.6M, 0.85M, 1.0M, 1.25M, 1.5M, 1.7M, 1.85M, or 2.0M.

In various embodiments, the agent is citric acid, for example at 100 mM,or K₂HPO₄, for example at 50 mM at pH7.4.

According to further embodiments of the present invention, the Vacciniavirus is eluted with GAG or a GAG-like ligand or part thereof, with theGAG-binding domain of A27L or part thereof, and/or with an0-glycoside-binding cleaving enzyme.

In another embodiment, the agent is sodium chloride, more preferably, anincreasing NaCl concentration gradient ranging from 0.15 M to 2.0 M.

Pre-Treatment

Prior to loading on the solid phase, a pre-treatment of the virussuspension can be performed, specifically in order to removecontaminants from the Vaccinia virus in the liquid-phase culture.

Pre-treatment can be one or more of the following steps either alone orin combination:

1) Homogenization of the Host Cells

-   -   Ultrasound treatment    -   Freeze/thaw    -   Hypo-osmotic lysis    -   High-pressure treatment

2) Removal of Cell Debris

-   -   Centrifugation    -   Filtration

3) Removal/Reduction of Host Cell DNA

-   -   Benzonase treatment    -   Cationic exchange    -   Selective precipitation by cationic detergents

According to a further embodiment of the invention, the pH value of theviral suspension is decreased just prior to loading in order to improvethe binding of the virus particle to the ligand. The pH value of theviral suspension can be decreased from appr. pH 7.0-8.0 to 4.0-6.9, inparticular to pH 4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0, 5.2, 5.4, 5.5, 5.6,5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.8, 6.9. Preferably, the pH value isdecreased from pH 7.0-8.0 to pH 5.8. Subsequently, just after loadingand before elution, the pH value is again increased to pH 7.0-8.0, inparticular to pH 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, 8.0, preferably to pH7.7, in order to improve the stability of the viral particles.

Post-Treatment

Depending on the agent used for elution of the Vaccinia virus orrecombinant virus, post-treatment can be performed to enhance the purityof the virus preparation. The post-treatment could beultra/diafiltration for further removal of impurities and/or specific ornon-specific agents used for elution. To obtain an efficientpurification of the virus, it is also preferred to combine thepurification according to the invention with one or more furtherpurification steps, e.g., by ion-exchange(s). Ion-exchange(s) can, then,also be performed as post-treatment step(s).

In order to prevent aggregation of the purified virus suspension and,thus, to, inter alia, improve the detection of infectious particles, inparticular by the TCID₅₀ method, it can also be suitable to increase thepH value after elution of the virus, in particular to a pH value of upto 9 or more, in particular to pH 7.5, 7.6, 7.8, 8.0, 8.2, 8.4, 8.5,8.6, 8.8, 9.0, 9.2, 9.4, 9.5, 9.6, 9.8, 10.0, 10.2, 10.4, 10.5.Preferably, the pH value is increased from, in particular, pH 7.0, 7.2,7.4, 7.5, 7.6, 7.8, 8.0, preferably pH 7.7 to pH 9.0.

Preferably, the amount of host-cell DNA in a VV dose of 1×10⁸ TCID₅₀ is10-20 μg, 1-10 μg, 100 ng-1 μg, 10-100 ng, or 1-10 ng. In variousembodiments, the amount of host-cell DNA is less than 100 ng, 50 ng, 20ng, 10 ng, 5 ng, or 1 ng per ml or less than 100 ng, 50 ng, 20 ng, 10ng, 5 ng, or 1 ng. The amount of dsDNA in a VV sample can be reduced bythe purification method to less than 40%, 20%, 10%, 5%, 2.5, 1%, 0.5%,0.25%, 0.1%, 0.05%, 0.02% or 0.01% of input.

In various embodiments, the amount of protein in the purified VV is lessthan 250 μg/ml, 100 μg/ml, 50 μg/ml, 20 μg/ml, 10 μg/ml, or 5 μg/ml. Invarious embodiments, the amount of protein in the purified VV is lessthan 250 μg/1×10⁸ TCID₅₀, 100 μg/1×10⁸ TCID₅₀, 50 μg/1×10⁸ TCID₅₀, 20μg/1×10⁸ TCID₅₀, 10 μg/1×10⁸ TCID₅₀, or 5 μg/1×10⁸ TCID₅₀. The amount ofcontaminating protein is preferably less than 40%, 20%, 10%, 5%, 2.5,1%, 0.5%, 0.25%, 0.1%, 0.05%, 0.02% or 0.01% of input.

The practice of the invention employs techniques in molecular biology,protein analysis, and microbiology, which are within the skilledpractitioner of the art. Such techniques are explained fully in, forexample, Ausubel et al. 1995, eds, Current Protocols in MolecularBiology, John Wiley & Sons, New York.

Modifications and variations of this invention will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by the way of example only, and the invention is not to beconstrued as limited thereby. Additional aspects and advantages of theinvention will be set forth in part in the description which follows,and in part will be obvious from the description, or may be learned bypractice of the invention.

In one embodiment, the invention provides a more time-effective andcost-effective process for purification of Vaccinia viruses andrecombinant-modified Vaccinia viruses in higher yield, comprising one ormore of the following steps:

a. loading a solid-phase matrix with a liquid-phase virus preparation,wherein the solid-phase matrix comprises a ligand appropriate forinteracting with the virus, e.g. by reversibly binding the virus

b. washing of the matrix, and

c. eluting the virus.

In a preferred embodiment, the method comprises the following steps:

a. Loading a column, membrane, filter or similar solid-phase matrixcomprising one or more appropriate virus-binding ligands with aliquid-phase virus preparation,

b. Washing of the matrix with an appropriate solvent to removecontaminants, and

c. Eluting the Vaccinia virus with an appropriate solvent to achieve ahighly pure, biologically active, stable virus preparation.

In a further preferred embodiment, the method comprises the followingsteps:

a. Loading a column, membrane, filter or similar solid-phase matrixcomprising one or more appropriate HIC ligands with a liquid-phase viruspreparation

b. Washing of the matrix with an appropriate solvent to removecontaminants, and

c. Eluting the Vaccinia virus with citric acid, for example at 100 mM,or K₂HPO₄, for example at 50 mM at pH7.4, to achieve a highly pure,biologically active, stable virus preparation.

In one particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

a. Loading a column, membrane, filter or similar solid-phase HIC matrixsubstituted with a phenyl or PPG group with a Vaccinia virus preparationdissolved in a neutral buffer (pH 6.5 to 8.5, preferably >=pH 7.5) inammonium sulphate, preferably at 1.5-1.7M,

b. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, and

c. Eluting the Vaccinia virus with citric acid, for example at 100 mM,or K₂HPO₄, for example at 50 mM at pH7.4 to obtain the biologicallyactive Vaccinia virus particles.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

a. Loading a column, membrane, filter or similar solid-phase matrixsubstituted with a Heparin (HP) with a Vaccinia virus preparationdissolved in a neutral buffer (pH 6.5 to 8.5, preferably >=pH 7.5) witha physiological salt concentration (approximately 150 mM NaCl). Anappropriate buffer is Phosphate Buffered Saline (PBS), e.g. 0.01 to 0.1M phosphate, 0.15 M NaCl, pH 7.5. Other appropriate buffers areTris-NaCl, e.g. 0.01 to 0.1 M Tris, 0.15 M, or citric acid, at 100 mM,or K₂HPO₄ at 50 mM at pH7.4.

b. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, for example, as measured by the return ofthe 280 nm absorbance signal to the pre-loading baseline, and

c. Eluting the Vaccinia virus with 1.5-1.7M ammonium sulphate containing50 mM K₂HPO₄ at pH7.4.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

a. Loading a column, membrane, filter or similar solid-phase sulphatedcellulose matrix with a Vaccinia virus preparation dissolved in aneutral buffer (pH 6.5 to 8.5, preferably >=pH 7.5) with a physiologicalsalt concentration (approximately 150 mM NaCl). An appropriate buffer isPhosphate Buffered Saline (PBS), e.g. 0.01 to 0.1 M phosphate, 0.15 MNaCl, pH 7.5. Other appropriate buffers are Tris-NaCl, e.g. 0.01 to 0.1M Tris, 0.15 M, or citric acid, at 100 mM, or K₂HPO₄ at 50 mM at pH7.4.

b. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, for example, as measured by the return ofthe 280 nm absorbance signal to the pre-loading baseline, and

c. Eluting the Vaccinia virus with 1.5-1.7M ammonium sulphate containing50 mM K₂HPO₄ at pH7.4.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

a. Loading a column, membrane, filter or similar solid-phase matrixsubstituted with a Heparin (HP) with a Vaccinia virus preparationdissolved in a neutral buffer (pH 6.5 to 8.5, preferably >=pH 7.5) witha physiological salt concentration (approximately 150 mM NaCl). Anappropriate buffer is Phosphate Buffered Saline (PBS), e.g. 0.01 to 0.1M phosphate, 0.15 M NaCl, pH 7.5. Other appropriate buffers areTris-NaCl, e.g. 0.01 to 0.1 M Tris, 0.15 M, or citric acid, at 100 mM,or K₂HPO₄ at 50 mM at pH7.4.

b. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, for example, as measured by the return ofthe 280 nm absorbance signal to the pre-loading baseline,

c. Eluting the Vaccinia virus with 1.5-1.7M ammonium sulphate containing50 mM K₂HPO₄ at pH7.4,

d. Loading a column, membrane, filter or similar solid-phase HIC matrixwith the eluted Vaccinia virus preparation,

e. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, and

f. Eluting the Vaccinia virus with citric acid, for example at 100 mM,or K₂HPO₄, for example at 50 mM at pH7.4 to obtain the biologicallyactive Vaccinia virus particles.

In another particularly preferred embodiment, the method is used for thepurification of biologically active Vaccinia virus and comprises thefollowing steps:

a. Loading a column, membrane, filter or similar solid-phase sulphatedcellulose matrix with a Vaccinia virus preparation dissolved in aneutral buffer (pH 6.5 to 8.5, preferably >=pH 7.5) with a physiologicalsalt concentration (approximately 150 mM NaCl). An appropriate buffer isPhosphate Buffered Saline (PBS), e.g. 0.01 to 0.1 M phosphate, 0.15 MNaCl, pH 7.5. Other appropriate buffers are Tris-NaCl, e.g. 0.01 to 0.1M Tris, 0.15 M, or citric acid, at 100 mM, or K₂HPO₄ at 50 mM at pH7.4.

b. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, for example, as measured by the return ofthe 280 nm absorbance signal to the pre-loading baseline, and

c. Eluting the Vaccinia virus with 1.5-1.7M ammonium sulphate containing50 mM K₂HPO₄ at pH7.4,

d. Loading a column, membrane, filter or similar solid-phase HIC matrixwith the eluted Vaccinia virus preparation,

e. Washing of the matrix with a sufficient amount of the loading bufferto ensure complete elution of all non-binding Vaccinia virus particlesand non-binding contaminants, and

f. Eluting the Vaccinia virus with citric acid, for example at 100 mM,or K₂HPO₄, for example at 50 mM at pH7.4 to obtain the biologicallyactive Vaccinia virus particles.

EXAMPLE 1 Production of Modified Vaccinia Ankara Virus Particles

MVA-BN® virus particles were produced by Bavarian Nordic A/S (Denmark)in primary cultures of CEF cells under Good Manufacturing Practiceconditions (Vollmar et al., 2006). Different batches of the startingmaterial were provided after homogenization and clarification as aliquid frozen product, stored in aliquots at −80° C. The initial TCID₅₀values of the samples were calculated.

EXAMPLE 2 Total Protein Assay

Total protein concentrations were determined in triplicates by thePierce® BCA protein assay reagent kit (Cat.#23225, Pierce Biotechnology,USA) as recently described (Wolff et al. 2009). The assay was calibratedagainst albumin standards (BSA) (Cat.#23209, Thermo Fisher ScientificInc., USA) within the validated working range of 25 to 250 μg/ml (limitof detection: 8.3 μg/ml; limit of quantification: 25 μg/ml).

EXAMPLE 3 Quantification of MVA-BN® Virus Particles

Total MVA-BN® virus particles were quantified in triplicates by asandwich ELISA as described previously (Wolff et al. 2009).

Infectious MVA-BN® virus particles were determined by the 50% tissueculture infective dose assay (TCID₅₀) in Vero cells (ECACC;Cat.#88020401, UK; 2.0×10⁵ cells/well) as a variation of the proceduredescribed by Jordan et al. (Jordan et al. 2009). Briefly, Vero cellswere maintained in high glucose (4.5 g/l) DMEM-medium (Cat.# E15-009,PAA Laboratories GmbH, Cölbe, Germany) containing 4 mM glutamin (Cat.#G-3126-250G Sigma-Aldrich, München, Germany), 0.1% gentamycin(Cat.#15710080, Invitrogen, Karlsruhe, Germany) and 10% FBS(Cat.#3302-P280703, PAN-Biotech GmbH, Aidenbach, Germany) at 37° C. and5% CO₂. Serial 10-fold dilutions of the virus containing samples wereadded to Vero monolayers. After incubation (48 h) the cells were fixedwith a 1:2 acetone (Cat.# CP40.3, Carl Roth, Karlsuhe, Germany):methanol (Cat.#106018, Merck, Darmstadt, Germany) mixture and incubatedwith a polyclonal rabbit anit-vaccinia virus antibody (Cat.#220100717,Quartett Immunodiagnostika & Biotechnologie GmbH, Berlin, Germany) at1:1000 dilution in PBS containing 1% FBS. Subsequently, the wells werewashed with PBS and incubated with the secondary antibody (anit-rabbitIgG, peroxidase conjugated, Cat.# W401B, Promega GmbH, Mannheim,Germany) in PBS containing 1% FBS. The peroxidase enzyme of thesecondary antibody catalyses a color reaction upon incubation with ACEsubstrate solution (0.3 mg/ml 3-amino-9-ethyl-carbozole (Cat.#A5754-10G, Sigma-Aldrich, München, Germany) in 0.1 M Na-acetate bufferpH 5.0 containing 0.015% H₂O₂ (Cat.#1.07209.0250, Merck, Darmstadt,Germany). Infected forci were identified under the light microscope andthe TCID₅₀ is calculated from the maximum dilution of MVA-BN® suspensionthat yields positive dye reaction. All titrations were performed inparallel replicates.

EXAMPLE 4 DNA-Quantification Assays

The dsDNA measurements in a validated working range from 4 to 1000 ng/mlwere done as described by Opitz et al. (Opitz et al. 2007) using theQuant-iT™ PicoGreen® dsDNA reagent from Molecular Probes, Inc. (Cat.#P7581, Eugene, Oreg., USA). Calibration was done against lambda DNA(Cat.# D1501, Promega Corporation, Madison, Wis., USA) within thevalidated working range of 4 to 1000 ng/ml (weighted regression; limitof detection: 0.66 ng/ml; limit of quantification: 2.36 ng/ml) using 100mM citric acid buffer pH 7.2 for dilutions.

Total DNA measurements in a range of 6 to 400 pg/ml were done by thetotal DNA Threshold assay method after protease treatment and DNAextraction.

EXAMPLE 5 Protease Treatment

Samples were dialysed (5000 kDa MWCO; Cat.#131192, Sectrum Europe B.V.,Breda, Netherlands) against 50 mM phosphate buffered saline containing 1mM EDTA and 0.05% NaN₃, pH 7.0 and appropriately diluted in the ZeroCalibrator solution (50 mM PBS, 1 mM EDTA, 0.05% NaN₃, pH 7.0; Cat.# R8004, MDS Analytical Technologies, Ismaningen, Germany) containing 0.01mg/ml SDS (Cat.# L 6026, Sigma-Aldrich, München, Germany) and 0.01 mg/mlProteinase K (Cat.# P8102S, New England Biolabs GmbH, Frankfurt,Germany) and incubated over night at 56° C.

EXAMPLE 6 DNA Extraction

The DNA was extracted after protease treatment using a DNA extractor kit(Cat.#295-58501, Wako Chemicals GmbH, Neuss, Germany) according to themanufactures instructions.

EXAMPLE 7 DNA Quantification

DNA Quantification was done by the Threshold Total DNA Assay Kit (Cat.#R 9009, MDS Analytical Technologies, Ismaning, Germany) and workstation(MDS Analytical Technologies, Ismaning, Germany) as described in thefollowing. After extraction, samples were adjusted to 500 μl with zerocalibrator solution and heat denatured at 105° C. for 15 min. 1000 μl ofa mixture containing biotin-conjugated, high affinity, single-strandedDNA binding protein, streptavidine and urease-conjugated monoclonalantibody against single-stranded DNA (ssDNA) were added to each sampleor standard and incubated for 1 hour at 37° C. The reaction mixtureswere transferred to individual wells in the manifold of the Thresholdworkstation. Mixtures were filtered through the biotin-coatednitrocellulose membrane adsorber under controlled vacuum. Subsequently,the wells were washed (wash solution, phosphate buffered saline, pH 6.5containing 0.05% NaN₃ and 0.05% Tween 20) and the filtration wascontinued under high vacuum until all wells were dry. Then the dipstickmembrane adsorbers were transferred to the Threshold reader whichcontained the substrate urea (600 μl of 5 M urea containing 0.05% NaN₃and 30 μl wash solution) and the light-addressable potentiometricsensor. Captured urease in the DNA-protein complexes hydrolyses ureawhich results in detectable pH changes in the substrate solution. Allsamples were measured in triplicates. The assay was calibrated againstcalf thymus DNA and all samples were analyzed additionally by spikingthem with (50 pg) calf thymus DNA in order to estimate spike recoveriesaccording to the manufacturer's recommendations. All the controls werewithin the range indicated on the certificate of analysis from thesupplier.

EXAMPLE 8 Chromatography Materials

Pseudo-Affinity Membrane Adsorbers—

Heparin-MA was a research product of Sartorius Stedim Biotech GmbH,Göttingen, Germany. It was based on reinforced stabilized cellulose witha pore size >3 μm and adsorption area of 3×75 cm² by 3×15 layers. Thehousing material was polypropylene. Sulfated cellulose MA (SC-MA) with adiameter of 25 mm (pore size >3 μm, Sartorius Stedim Biotech GmbH,Göttingen, Germany) were prepared as described previously (Opitz et al.2009), except that the membrane discs were incubated for 12 hours at 35°C., 40° C. and 45° C. The adsorption area was 75 cm², and 15 membranediscs were stacked in a stainless steel membrane holder (Cat.#1980-002,GE Healthcare, München, Germany). Membrane adsorbers prepared at 40° C.have been applied for the majority of experiments, other membranes wereused to optimize the sulfation degree in terms of dynamic bindingcapacity and purity. Sulfate ion content of blank and modified sulfatedcellulose MA was estimated by the Schöniger decomposition methodfollowed by ion exchange chromatography (Currenta GmbH & Co. OHG,Leverkusen, Germany)

Hydrophobic Interaction Chromatography Matrices—

Experiments were done with 1 ml columns of the ToyoScreen HIC Mix Pack(Cat.#21398, Tosoh Bioscience GmbH, Stuttgart, Germany). The screenedresins comprised ToyoScreen® Hexyl-650C, ToyoScreen® Butyl-600M,ToyoScreen® Phenyl-650M, ToyoScreen® PPG-600M, ToyoScreen® Ether 650M.

EXAMPLE 9 Adsorption Chromatography

Chromatography was performed using an Äkta Explorer system (GEHealthcare, München, Germany) at a flow rate of 1.0 ml/min and monitoredby UV (280 nm) and light scattering (90°, Dawn EOS, Wyatt TechnologyEurope GmbH, Dernbach, Deutschland) detection.

Dynamic binding capacity of the HIC-chromatography media was determinedloading the clarified MVA-BN® virus sample (1.85×10⁸ TCID₅₀/ml) inadsorption buffer (1.7 M (NH₄)₂SO₄+50 mM K₂HPO₄, pH 7.4; HIC (1.7)) ontoequilibrated (HIC (1.7) adsorption buffer) 1 ml columns of theHIC-columns. The breakthrough was monitored via light scatteringdetector and the virus particles were eluted with 50 mM K₂HPO₄, pH 7.4.

Dynamic binding capacities of the pseudo-affinity MA (SC-MA andheparin-MA) were determined loading the clarified MVA-BN® virus sample(1.85×10⁸ and 4.65×10⁷ TCID₅₀/ml) in SC-MA adsorption buffer (100 mMcitric acid, pH 7.4) at a flow rate of 1 ml/min onto equilibrated (SC-MAadsorption buffer) 75 cm² SC-MA and heparin-MA. The breakthrough wasmonitored via light scattering detector and the virus particles wereeluted with 100 mM citric acid containing 2 M NaCl, pH 7.4. The elutedproduct fraction was dialysed against adsorption buffer with a MWCO of5000 kDa (Cat.#131192, Spectrum Europe B.V., Breda, Netherlands) and thevirus and DNA content was quantified as described above.

Characterization of the HIC-materials was done with 4 ml of theclarified MVA-BN® virus sample (4.65×10⁷ TCID₅₀/ml) in HIC (1.7) and HIC(1.5; 1.5 M (NH₄)₂SO₄+50 mM K₂HPO₄, pH 7.4) adsorption buffer. Prior tosample loading the chromatography material was equilibrated with therespective HIC adsorption buffers. After a brief washing (respective HICadsorption buffer) the bound virus particles were eluted with elutionbuffer (50 mM K₂HPO₄, pH 7.4). Resulting fractions were pooled andanalyzed for virus and contaminant compositions. Chromatographicmaterials were regenerated after each run with 10 column volumes of 0.5M NaOH and 0.1 M HCl. All experiments were performed in triplicates.

Optimization of the Ammonium Sulfate Concentration for the MVA-BN®Adsorption onto HIC-Phenyl Resin

The study was done as the characterization of the different HIC-matricesdescribed above. However, the HIC-adsorption buffer for the sampleloading and column equilibration varied. The tested adsorption bufferscontained 0.45, 0.6, 0.85, 1.0, 1.25, 1.5 and 1.7 M (NH₄)₂SO₄ and 50 mMK₂HPO₄, pH 7.4.

Combination of Pseudo-Affinity MA and HIC

The chromatography was performed using the same system and monitored asdescribed above at a flow rate of 1.0 ml/min. Four ml of the clarifiedMVA-BN® virus sample (4.65×10⁷ TCID₅₀/ml) in 100 mM citric acid, pH 7.4or 50 mM K₂HPO₄, pH 7.4 have been subjected to an equilibrated (100 mMcitric acid, pH 7.4 or 50 mM K₂HPO₄, pH 7.4) SC-MA (75 cm²) orheparin-MA (225 cm²). The virus was eluted from the pseudo-affinity MAafter a brief washing (100 mM citric acid, pH 7.4 or 50 mM K₂HPO₄, pH7.4) in HIC (1.5 and 1.7) adsorption buffer. The pooled eluted fractionswere directly loaded onto an equilibrated (respective HIC-adsorptionbuffer) ToyoScreen® Phenyl-650M or ToyoScreen® PPG-600M column. Theadsorbed virus particles were desorbed after washing (respectiveHIC-adsorption buffer) from the HIC-matrices with 50 mM K₂HPO₄ pH 7.4 or100 mM citric acid pH 7.4. Pooled fractions were stored at −80° C. Thevirus content and the amount of total dsDNA and protein were determinedfrom representative samples as described above. Analytical samplesremoved were considered in the overall mass balances.

Optimization of the Pseudo-Affinity Membrane Adsorbers

Table 1 demonstrates the dependence of the cellulose sulfation on thechemical reaction temperature. Reaction temperatures of 35, 40 and 45°C. resulted in 5.5, 9.3 and 13 weight % sulfation of the cellulosebackbone. The dynamic binding capacity up to 50 ml (9.3×10⁹ TCID₅₀)MVA-BN® was not affected by the degree of sulfation. However, theperformance in terms of product adsorption and DNA depletion variedamong the tested SC-MA. The SC-MA modified at the lowest reactiontemperature reflects the modest amount of adsorbed virus particles(66%). In contrast, SC-MA sulfated at 40° C. and 45° C. yielded aproduct recovery of 79% and 80%, respectively. The amount of total DNAin the product fraction increased with the degree of sulfation. Therelative amount of DNA based on the starting material in the product wasfor the SC-MA produced at 35° C., 40° C. and 45° C. 7.4%, 14% and 17%,respectively. The unmodified cellulose backbone bound 15% DNA whereas31% of the MVA-BN® virus particles adsorbed to it. Recent studiesdemonstrated the encouraging performance of pseudo-affinity MA based onsulfated cellulose compared to ion exchange MA (Wolff et al. 2009).These studies were conducted with MA sulfated at a reaction temperatureof 37° C. and suffered from losses (36%; (Wolff et al. 2009)) of virusparticles during the adsorption process.

Table 1 indicates that elevated cellulose sulfation lead within thetested temperature range to improved adsorption of the MVA-BN® virusparticles. The level of sulfation also seems to affect the DNAadsorption. Higher sulfated SC-MA resulted in enhanced adsorption oftotal DNA (Tab. 1). In contrast un-sulfated (<0.05 wt %) cellulose disksadsorbed 15% of the initial DNA content compared to 7.4% of the leastsulfated (5.5 wt %) SC-MA. This phenomenon can be explained by differentinteraction modes between sulfated and un-sulfated cellulose andDNA-molecules. The adsorption of DNA to hydrophilic surfaces likecellulose is commonly known and described in the literature as e.g. thepartial adsorption of nucleic acids to cellulose powder (Halder et al.2005) and the adsorption of non-circular DNA to a highly porouscellulose matrix (Deshmukh and Lali 2005). Enlarged DNA adsorption at anincreasing degree of sulfation is unexpected due to the ionic phosphategroups of nucleic acids. However, previous studies with CEF cell-derivedMVA-BN® virus particles displayed compared to anion exchange MA alimited adsorption of dsDNA to weak cation exchange MA and to thecationic pseudo-affinity MA like sulfated cellulose and heparin as wellas the bead-based sulfated cellulose resin Cellufine® sulfate (Wolff etal. 2009). Opitz et al. demonstrated similarly the adsorption of hostcell DNA during the primary capturing step of MDCK cell-derivedinfluenza virus particles to strong and weak cation exchange MA (Opitzet al. 2009).

Table 1: Effect of the sulfation degree from sulfated cellulose on thedynamic binding capacity, purity and overall virus yield. Relativeamounts (mean and standard deviation of triplicates) for MVA-BN® (ELISA)and dsDNA (Quant-iT® PicoGreen® assay) content were calculated based onthe starting material of the homogenized and clarified virus broth. Theadsorption area of the SC-MA was 75 cm₂. Equilibration and wash bufferwas 100 mM citric acid, pH 7.4, and the elution buffer 100 mM citricacid+2 M NaCl, pH 7.4. The product recoveries from the cellulosebackbone (blank) and the sulfated cellulose MA were estimated from 2chromatographic experiments. The dynamic binding capacity experimentswere done twice.

TABLE 1 Dynamic Recoveries in Chroma- Sul- Binding Capacity ProductFraction tography fation Volume Total TCID₅₀ MVA-BN ® Total Media [wt %][ml] [TCID₅₀ ] [%] DNA [%] Cellulose <0.05 n. d. n. d. 31 ± 0.2  15 ±0.4 backbone SC-MA 5.5 >50 >9.3 × 10⁹ 66 ± 3.3 7.4 ± 1.5 35° C. SC-MA9.3 >50 >9.3 × 10⁹ 79 ± 2.4  14 ± 0.6 40° C. SC-MA 13 >50 >9.3 × 10⁹ 80± 6.7  17 ± 0.5 45° C.

Dynamic Binding Capacities of the Tested HIC-Resins and Pseudo-AffinityMA

Table 2 shows the dynamic binding capacities of the testedchromatography materials. The capacity of all tested HIC resins wasgreater than 20 ml of the homogenized and clarified harvest (3.7×10⁹TCID₅₀). After 20 ml the addition of MVA-BN® virus sample was stopped,because the dynamic binding capacity was judged sufficient for thecharacterization of the HIC columns. The capacity of the heparin-MA was6.0 ml corresponding to 1.1×10⁹ TCID₅₀, and the capacity of the SC-MA asalready discussed was independent of the degree of sulfation greaterthan 50 ml (9.3×10⁹ TCID₅₀; Tab. 1). The high dynamic binding capacityfor the SC-MA was verified via quantification of the viral particlesafter elution from the SC-MA and compared with data obtained during thecharacterization of the SC-MA. The recovered virus particles based onthe loaded sample for 50 ml and 4 ml were 81% and 79%, respectively. Theun-adsorbed virus particles were for the respective experiments 22% and23%. Thus, it can be assumed that the virus particles of 9.3×10⁹ TCID₅₀did adsorb to the MA and filtration effects at a pore size of 3 to 5 μm,if at all, are negligible. Furthermore, these experiments confirm thatnon-specific binding to the chromatography materials at the selectedvolume for the characterization studies (4 ml) were insignificant.Loading of the MVA-BN® virus sample during the dynamic binding capacitystudies was stopped after 50 ml to conserve sample. Earlier studiesdemonstrated the high dynamic binding capacity for Cellufine® sulfate, acommercial bead based resin constituted of sulfated cellulose beads,supporting the observed high capacity of SC-MA (Wolff et al. 2009).

Table 2: Dynamic binding capacity of the tested chromatographymaterials. The adsorption buffer for the hydrophobic interactionchromatography media (1 ml column) was 1.7 M (NH₄)₂SO₄+50 mM K₂HPO₄, pH7.4 and for the pseudo-affinity membrane adsorbers (gray; adsorptionarea: 75 cm²) 100 mM citric acid, pH 7.4.

TABLE 2

Screening of HIC Resins

Preliminary studies with NaCl (2 M), an intermediate chaotropic salt didnot lead to sufficient adsorption of virus particles or nucleic acids(data not shown) to ethyl, phenyl and hexyl ligands. Hence, thesuitability of HIC for the depletion of contaminating DNA afterpseudo-affinity chromatography was evaluated during this study with astrong antichaotropic salt, ammonium sulfate, with a series of differenthydrophobic ligands. Selection of the most promising HIC-ligands wasconducted in experiments comprising the following ligands: ether,poly-propylene glycol (PPG), phenyl, butyl, and hexyl. The outcomes ofthese experiments are combined in FIG. 1. The majority of MVA-BN® virusparticles adsorbed to the tested HIC-resins. For the PPG and phenylligand, no virus particles were detected via ELISA in the flow throughfraction under the applied conditions. In case of the ether, butyl andhexyl HIC-ligands 3%, 6% and 7%, respectively, of the initial amount ofvirus were detected in the flow through fraction. However, the overallmaterial balances for the MVA-BN® virus particles could not be closedand relative amounts of virus detected in the product fraction rangedfrom 55% (ether) to 88% (PPG). For phenyl, butyl and hexyl ligands 84%,67% and 63%, respectively, of the initial amount of virus were measuredin the product fraction.

The fraction of un-adsorbed DNA varied for the tested HIC-resins withether (75%), PPG (64%), phenyl (58%), butyl (48%) and hexyl (53%; FIG.1). The amount of co-eluted DNA with virus particles was for thedifferent HIC-ligands as ether (29%), PPG (13%), phenyl (19%) and forbutyl and hexyl 4%. Here, an increase in hydrophobicity with growingn-alkyl chain length (Queiroz et al. 2001) lead to an elevated portionof strong bound DNA, resulting in a reduced overall recovery of DNA. Forthe more hydrophobic ligands like butyl and hexyl 48% and 43% of theinitial DNA content could not be accounted for in the material balances.Strong bound DNA were presumably removed from the HIC-resins during theregeneration step. The hydrophobic character as confirmed in theseexperiments is frequently exploited for the purification of plasmid DNA(Diogo et al. 2001; Diogo et al. 2005; Freitas et al. 2009; Iuliano etal. 2002). These applications benefit from the relatively highhydrophobicity of genomic DNA due to the exposure of the hydrophobicbases, compared to plasmid DNA molecules, where the majority of thebases are shielded inside the double helix (Freitas et al. 2009). Thepresented results clearly reveals that the hydrophobic character of freeDNA from the MVA-BN® cultivation broth can be utilized to removeresidual DNA from MVA-BN® virus particles after pseudo-affinitychromatography.

Proteins were heavily adsorbed to all tested HIC-resins under the testedconditions. Except for the ether ligand, were 2% of the initial proteincontent did not adsorb to the resin, no proteins were determined in theflow through for all other HIC-materials. The applied elution conditionsin absence of ammonium sulfate, was not sufficient to desorb theproteins from the HIC-resins. The amount of total protein in the productfraction ranged from the detection limit (butyl, hexyl) to 2% (PPG). Theproduct fractions of the ether and phenyl HIC-ligands contained 1% totalprotein. The majority of remaining proteins on the matrix were removedfrom the HIC-adsorbers during the harsh regeneration procedure. Anyeffects on dynamic capacity losses have not been observed within thetested range during the studies. However, if HIC adsorbers are appliedas an orthogonal purification step to the pseudo-affinity membraneadsorbers, the majority of proteins are already removed and the overallcapacity of the HIC-resins will not be significantly affected by theremaining protein load.

The high MVA-BN® recovery and DNA depletion of the PPG and phenyl HICresins lead to the selection of these resins to explore theirperformance for a MVA-BN® vaccine downstream process in combination withan upstream pseudo-affinity chromatography.

Studies Combining Pseudo-Affinity MA and HIC

Table 3 illustrates the amount of MVA-BN® virus particles and DNA in theproduct fraction relative to the loaded sample. These experiments wereconducted with 2 different batches of MVA-BN® virus (batch A and B)under different buffer conditions. Here, virus particles were adsorbedand eluted from the chromatography materials via potassium phosphate orcitric acid buffers containing ammonium sulfate according to therespective studies. The applied chromatography media were sulfatedcellulose- and heparin-MA (pseudo-affinity MA) and the HIC-phenyl andHIC-PPG resins. Studies conducted with batch A involved any possiblecombination of the two different pseudo-affinity and HIC adsorptionmedia (Table 3).

Table 3: Purification of two different batches (A and B) of MVA-BN®virus particles by a sequential combination of pseudo-affinity membraneadsorbers (sulfated cellulose (SC-MA) and heparin (heparin-MA; grayhighlighted)) and 1 ml hydrophobic interaction chromatography columns(Phenyl and PPG). Relative amounts (mean and standard deviation oftriplicates) for MVA-BN® (ELISA), dsDNA content (Quant-iT® PicoGreen®assay) were calculated based on the starting material of the homogenizedand clarified virus broth. Total DNA amounts labeled by a star weredetermined by the Threshold system in place of the Quant-iT® PicoGreen®assay. The adsorption areas of the SC-MA and heparin-MA were 75 cm2 and225 cm², respectively. Pseudo-affinity equilibration and wash buffer was100 mM citric acid, pH 7.4 or 50 mM potassium phosphate buffer, pH 7.4as stated in the table, the pseudo-affinity elution buffer correspondedthe HIC-adsorption buffer (1.7 M (NH₄)₂SO₄, pH 7.4) and the HIC elutionbuffers were 50 mM K₂HPO₄, pH7.4 or 100 mM citric acid, pH 7.4.Individual chromatographic runs were done in triplicates from which themeans and standard deviations were calculated.

TABLE 3 Recoveries in Product Fraction Batch B Batch A MVA-BN ® TotalMVA-BN ® [%] Total DNA [%] [%] DNA [%] Chromatography Citric CitricCitric Citric Medium K₂HPO₄ acid K₂HPO₄ acid acid acid SC-MA 73 ± 1.7 75± 1.6 5.8 ± 3.9 4.9 ± 0.6 81 ± 3.1 10 ± 0.4 HIC-Phenyl 76 ± 0.5 74 ± 5.1 0.9 ± 0.4^(b)  0.2 ± 0.0^(b) 94 ± 1.1 5.6 ± 1.4  Overall recovery 55 560.04 0.01 76 0.6 Heparin-MA 68 ± 4.2 68 ± 0.6  12 ± 4.1  20 ± 0.7 62 ±1.7 19 ± 2.8 HIC-Phenyl 73 ± 1.1 76 ± 1.9  0.3 ± 0.2^(b)  0.3 ± 0.0^(b)71 ± 4.1 13 ± 1.9 Overall recovery 50 50 0.04 0.06 44 2.5 SC-MA 77 ± 5.671 ± 2.7 2.0 ± 0.6 4.2 ± 0.6 n. d. n. d. HIC-PPG 64 ± 1.8 62 ± 1.7LOQ^(a) LOQ^(a) n. d. n. d. Overall recovery 49 44 LOQ^(a) LOQ^(a) n. d.n. d. Heparin-MA 71 ± 2.4 59 ± 2.8  14 ± 0.5  20 ± 0.5 n. d. n. d.HIC-PPG 47 ± 1.4 60 ± 2.6 LOQ^(a) LOQ^(a) n. d. n. d. Overall recovery33 35 LOQ^(a) LOQ^(a) n. d. n. d. ^(a)limit of quantification, totalprotein concentration of all samples has been below the quantificationlimit; ^(b)determined via DNA Threshold assay

Final desorptions of the MVA-BN® product were done for all studies withpotassium phosphate and citric acid buffers as described above.

Potential batch to batch variations were briefly explored by theapplication of a second batch (batch B) of MVA-BN® virus particles.Combinations with the HIC-PPG columns were not carried out on grounds ofthe low virus recoveries for the studies with batch A. Furthermore,final desorptions were done only with citric acid buffer as nodifferences between the potassium phosphate and citric acid buffer wasobserved in the initial studies and citric acid may be beneficial toreduce potential virus aggregations due to the high negative charge atneutral pH. The bulk of MVA-BN® virus in the product fraction afterSC-MA chromatography ranged for experiments with potassium phosphate andcitric acid from 73% to 77% and 75% to 71%, respectively. The amount oftotal DNA varied for the potassium phosphate and citric acid experimentsfrom 5.8% to 2.0% and 4.9% to 4.2%, respectively. Hence, no significantdifferences between the individual set of experiments were encountered.However, virus yields were improved compared to previous reports (65%;(Wolff et al. 2009) as already discussed. DNA depletions were comparableto the previously reported values (Wolff et al. 2009).

Observations from the chromatographic performance of sulfated cellulosechromatography media like SC-MA or bead based sulfated cellulose(Cellufine® sulfate) for cell culture-derived influenza virus particles(Opitz et al. 2009) and MVA-BN® (Wolff et al. 2009) supporting thedescribed results. The quantity of MVA-BN® virus in the product fractionafter heparin-MA chromatography ranged for the experiments withpotassium phosphate and citric acid from 68% to 71% and 59% to 68%,respectively. The amount of total DNA varied for the potassium phosphateexperiments from 12% to 14% and for both sets of the citric acidexperiments 20% were co-eluted with the product.

Virus recoveries after loading the homogenized and clarified harvestonto the HIC-PPG and HIC-phenyl columns differed significantly from therecoveries after subjecting the pseudo-affinity chromatography processedsamples over the same HIC columns. The MVA-BN® virus recoveries for thehomogenized harvest were 88% and 84% for the HIC-PPG and HIC-phenylcolumn, respectively. On the contrary, average virus recoveries for thepseudo-affinity chromatography purified samples achieved over all fourHIC-PPG and HIC-phenyl experimental series were 58% and 75%,respectively. The increased losses are expected due to the differencesin sample load and here in particular due to the heavily reduced proteinload after pseudo-affinity chromatography, which could influence theadsorption behavior of the remaining virus particles.

As expected from the results of the individual unit operations, thebuffer systems did not impact heavily the overall virus recoveries.Focusing on the citric acid buffered experiments optimal virus yieldswere accomplished with the SC-MA/HIC-phenyl combination (56%) followedby the heparin-MA/HIC-phenyl (50%), SC-MA/HIC-PPG (44%) and theheparin-MA/HIC-PPG (35%; Tab. 3). Due to the low overall virusrecoveries for the HIC-PPG combinations residual DNA levels were onlytested via the PicoGreen® assay and not further characterized via theThreshold assay system. Final DNA amounts in the product fractionsvaried for the SC-MA/HIC-phenyl combination insignificantly between0.04% and 0.01% of the starting material (Threshold assay, Tab. 3).

However, initial tests exploring batch to batch variations by repeatingsome of the experiments with batch B of homogenized and clarifiedharvest resulted in an increased residual DNA content in the productfraction in particular after the HIC-phenyl chromatography. The DNAcontent in the final product fraction was for the SC-MA/HIC-phenyl andthe heparin-MA/HIC-phenyl combination using virus batch B 0.6% and 2.5%(Tab. 3), respectively. While the DNA content after the pseudo-affinitychromatography using the heparin-MA was comparable, for the SC-MA therewas a two-fold increase. Main differences arised from the HIC-phenylstep, resulting in a 30 to 40-fold increase of the final DNA content inbatch B compared to batch A (Tab. 3).

Absolute amounts of total DNA were higher in the virus harvest for batchA than for batch B. Hence, it is not likely that capacity limitations ofthe HIC-adsorber could have lead to increasing residual DNA in theproduct fraction from batch B. Structural changes on the DNA-moleculesmay lead to these batch to batch variations. The integrity of DNAmolecules during the production process is mainly susceptible tocellular nucleases and shear, leading to fragmentation or structuralchanges. The activity and amount of free cellular nucleases depends onthe host cell viability during the final stages of the cultivation,which frequently varies. Shear stress should not vary heavily during theproduction process in the bioreactor. However, during the harvesting andclearance filtration this could potentially vary and shear induced DNAfragmentation is commonly known and has been described in severalpublications (Dancis 1978; Triyoso and Good 1999).

Overall virus yields varied between the two tested chromatographiccombinations. For the SC-MA/HIC-phenyl combination using citric acidcontaining buffers the virus yield was 56% (batch A) and 76% (batch B)and for the heparin-MA/HIC-phenyl arrangement 50% (batch A) and 44%(batch B). For the heparin-MA/HIC-phenyl downstream process the productyields from both unit operations (batch B) were slightly reduced leadingto an overall reduction of 6% compared to batch A. On the other hand,both unit operations of the SC-MA/HIC-phenyl set up resulted insignificant increased virus recoveries, leading to approximately 20%increased yield. The small variations between the different buffersystems and upstream applied pseudo-affinity MA for batch A (50% to 56%)compared to the virus recovery of 76% and 44% for batch B leads to theconclusion that the performance of both unit operations depict anoteworthy batch to batch variation which needs to be further exploredfor a routine application of the downstream process for MVA-BN® vaccineproducts. However, batch to batch variations of biotechnologicalproducts are common and need to be further addressed in processstability evaluations.

Protein concentrations were after both tested combinations ofpseudo-affinity and HIC-phenyl chromatography purifications below thequantification range of 25 μg/ml total protein (FIG. 2). After 10 foldconcentrations (lyophilization and buffer adaptation) of representativeHIC chromatography product fractions the limit for the quantificationrange was still not reached. The final protein concentration after thecharacterized combined purification steps was below 25 μg per dose.

Optimization of Ammonium Sulfate Concentration

The main function of the HIC was the further reduction of the DNAcontamination. This could be done in a positive or negative adsorptionmode or alternatively, via a differential elution of virus particles andDNA. The potential applicability of HIC-resins for this task was clearlydemonstrated by the combination of the pseudo-affinity MA and theHIC-phenyl resins (FIG. 3). Following studies focused for the mostpromising HIC-ligand (phenyl) on the reduction of the required ammoniumsulfate concentration to promote the adsorption of virus particles orDNA. At the concentration of 0.45 M ammonium sulfate 95% of the DNA and92% MVA-BN® virus particles did not adsorb to the HIC-phenyl resin.Increasing ammonium sulfate concentrations led to a sudden increase ofDNA adsorption of approximately 40% which was constant over the range oftested ammonium sulfate concentrations (0.6 M to 1.7 M).

The amount of DNA in the product fraction ranged from 10% to 20%.Ammonium sulfate concentrations larger than 0.45 M resulted in steadyincreasing virus adsorption. At 0.6 M, 0.85 M, 1.0 M, 1.25 M, 1.5 M and1.7 M ammonium sulfate about 43%, 58%, 63%, 77%, 85% and 85%,respectively, MVA-BN® virus particles were adsorbed and foundsubsequently in the product fraction. After 1.5 M ammonium sulfate novirus particles were detected in the flow through fraction. The DNAcontent in the eluted product fractions did not vary significantly atsalt concentrations applicable for virus adsorption and can therefore beneglected for the selection of the optimal ammonium sulfateconcentration of the adsorption buffer. The obtained results alsoclearly indicate that a differential elution of virus particles and DNAcan not be achieved by a partial reduction of the ammonium sulfateconcentration during the elution step. However, only roughly 40% of theDNA did adsorb to the HIC-phenyl resin at relevant ammonium sulfateconcentrations, from which approximately 20% could not be eluted underthe applied conditions for virus elution. Hence, the HIC phenyl resinrepresents a potential tool for a further DNA reduction by nearly 80%.The optimal salt concentration was judged based on the lowest possibleammonium sulfate concentration allowing complete virus adsorption (1.5 Mammonium sulfate). However, for reasons of process stability 1.7 Mammonium sulfate were used for the following studies. Comparing theresulting ammonium sulfate concentration with literature shows that formany different biomolecules ammonium sulfate concentrations of 1.5 to2.0 M are sufficient for high yield recoveries without denaturation(Kato et al. 2004). However, especially the denaturation aspect dependsmainly on the target and contaminating molecules.

Virus Infectivity after Downstream Processing

The effect of the described downstream process and especially the highammonium sulfate concentration on the virus infectivity was tested viathe TCID₅₀ assay. Therefore, representative samples were selected aftera SC-MA or heparin-MA and HIC-phenyl combination, with adsorptionbuffers containing 1.7 M ammonium sulfate. Each product fraction of thetriplicate chromatographic runs were assayed and the average TCID₅₀determined. The initial TCID₅₀ (blank for this particular assays) of thehomogenized and clarified harvest was 4.2×10⁷ for this particularexperiment. The average TCID₅₀ from the final product fractions of theSC-MA/HIC-phenyl, and heparin-MA/HIC-phenyl downstream processes were1.7×10⁷ and 1.6×10⁷ TCID₅₀, respectively. Hence, the entire processincluding HIC-adsorption, led to an approximate reduction of the TCID₅₀of 0.3 log units. The overall relative losses of the virus particlesbased on the ELISA quantification of the initially loaded sample were onaverage for both processes 47% (FIG. 2), which corresponds toapproximately 0.3 log units from the TCID₅₀. Therefore, it can beconcluded that losses on virus infectivity were not significantlyimpacted by the high concentration of ammonium sulfate.

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1. An industrial-scale method for the purification of biologicallyactive Vaccinia viruses comprising: i) binding the Vaccinia viruses to asolid-phase sulfated cellulose matrix; ii) eluting the Vaccinia viruses;iii) binding the eluted Vaccinia viruses to a solid-phase hydrophobicinteraction chromatograpy (HIC) matrix comprising a phenyl ligand; andiv) eluting a minimum of 5.0×10¹² Vaccinia virus particles.
 2. Themethod of claim 1, wherein the method is performed under asepticconditions.
 3. The method of claim 1, wherein the sulfated cellulosematrix comprises a sulfated reinforced cellulose membrane.
 4. The methodof claim 3, wherein the method is performed under aseptic conditions. 5.The method of claim 1, wherein the Vaccinia viruses are recombinantVaccinia virus.
 6. The method of claim 1, wherein the Vaccinia virusesare modified Vaccinia Ankara viruses or recombinant modified VacciniaAnkara viruses.
 7. The method of claim 6, wherein the method isperformed under aseptic conditions.
 8. The method of claim 1, whereinthe Vaccinia viruses are eluted from the sulfated cellulose matrix withammonium sulfate.
 9. The method of claim 8, wherein the Vaccinia virusesare eluted from the sulfated cellulose matrix with 1.7 M ammoniumsulfate.
 10. The method of claim 1, wherein the Vaccinia viruses areeluted from the HIC matrix with a citric acid buffer.
 11. The method ofclaim 1, further comprising a purification step by ion-exchange.
 12. Themethod of claim 11, wherein the method is performed under asepticconditions.
 13. The method of claim 2, further comprising administeringthe eluted Vaccinia virus to an animal.
 14. The method of claim 13,wherein the animal is a human.
 15. An industrial-scale method for thepurification of biologically active Vaccinia viruses comprising: i)binding the Vaccinia viruses to a solid-phase heparin ligand matrix; ii)eluting the Vaccinia viruses; iii) binding the eluted Vaccinia virusesto a solid-phase hydrophobic interaction chromatograpy (HIC) matrixcomprising a phenyl ligand; and iv) eluting a minimum of 5.0×10¹²Vaccinia virus particles.
 16. The method of claim 15, wherein the methodis performed under aseptic conditions.
 17. The method of claim 15,wherein the heparin ligand matrix comprises a heparin ligand membrane.18. The method of claim 17, wherein the method is performed underaseptic conditions.
 19. The method of claim 15, wherein the Vacciniaviruses are recombinant Vaccinia viruses.
 20. The method of claim 15,wherein the Vaccinia viruses are modified Vaccinia Ankara viruses orrecombinant modified Vaccinia Ankara viruses.
 21. The method of claim 1,wherein the sulfated cellulose matrix is a sulfated reinforced cellulosemembrane.
 22. The method of claim 15, wherein the heparin ligand matrixis a heparin ligand membrane.